Compounds for proteasome enzyme inhibition

ABSTRACT

Peptide-based compounds including heteroatom-containing, three-membered rings efficiently and selectively inhibit specific activities of N-terminal nucleophile (Ntn) hydrolases. The activities of those Ntn having multiple activities can be differentially inhibited by the compounds described. For example, the chymotrypsin-like activity of the 20S proteasome may be selectively inhibited with the inventive compounds. The peptide-based compounds include an epoxide or aziridine, and functionalization at the N-terminus. Among other therapeutic utilities, the peptide-based compounds are expected to display anti-inflammatory properties and inhibition of cell proliferation.

RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalPatent Application Ser. No. 60/599,401 filed on Aug. 6, 2004 and U.S.Provisional Patent Application Ser. No. 60/610,001 filed Sep. 14, 2004and is a continuation-in-part of U.S. patent application Ser. No.11/106,879 filed Apr. 14, 2005. The teachings of all of the referencedapplications are incorporated by reference in their entirety.

TECHNICAL FIELD

This invention relates to compounds and methods for enzyme inhibition.In particular, the invention relates to therapeutic methods based onenzyme inhibition.

BACKGROUND OF THE INVENTION

In eukaryotes, protein degradation is predominately mediated through theubiquitin pathway in which proteins targeted for destruction are ligatedto the 76 amino acid polypeptide ubiquitin. Once targeted, ubiquitinatedproteins then serve as substrates for the 26S proteasome, amulticatalytic protease, which cleaves proteins into short peptidesthrough the action of its three major proteolytic activities. Whilehaving a general function in intracellular protein turnover,proteasome-mediated degradation also plays a key role in many processessuch as major histocompatibility complex (MHC) class I presentation,apoptosis, cell growth regulation, NF-κB activation, antigen processing,and transduction of pro-inflammatory signals.

The 20S proteasome is a 700 kDa cylindrical-shaped multicatalyticprotease complex comprised of 28 subunits organized into four rings. Inyeast and other eukaryotes, 7 different a subunits form the outer ringsand 7 different β subunits comprise the inner rings. The α subunitsserve as binding sites for the 19S (PA700) and 11S (PA28) regulatorycomplexes, as well as a physical barrier for the inner proteolyticchamber formed by the two β subunit rings. Thus, in vivo, the proteasomeis believed to exist as a 26S particle (“the 26S proteasome”). In vivoexperiments have shown that inhibition of the 20S form of the proteasomecan be readily correlated to inhibition of 26S proteasome. Cleavage ofamino-terminal prosequences of β subunits during particle formationexpose amino-terminal threonine residues, which serve as the catalyticnucleophiles. The subunits responsible for catalytic activity inproteasomes thus possess an amino terminal nucleophilic residue, andthese subunits belong to the family of N-terminal nucleophile (Ntri)hydrolases (where the nucleophilic N-terminal residue is, for example,Cys, Ser, Thr, and other nucleophilic moieties). This family includes,for example, penicillin G acylase (PGA), penicillin V acylase (PVA),glutamine PRPP amidotransferase (GAT), and bacterialglycosylasparaginase. In addition to the ubiquitously expressed βsubunits, higher vertebrates also possess three interferon-γ-inducible βsubunits (LMP7, LMP2 and MECL1), which replace their normalcounterparts, X, Y and Z respectively, thus altering the catalyticactivities of the proteasome. Through the use of different peptidesubstrates, three major proteolytic activities have been defined for theeukaryote 20S proteasome: chymotrypsin-like activity (CT-L), whichcleaves after large hydrophobic residues; trypsin-like activity (T-L),which cleaves after basic residues; and peptidylglutamyl peptidehydrolyzing activity (PGPH), which cleaves after acidic residues. Twoadditional less characterized activities have also been ascribed to theproteasome: BrAAP activity, which cleaves after branched-chain aminoacids; and SNAAP activity, which cleaves after small neutral aminoacids. The major proteasome proteolytic activities appear to becontributed by different catalytic sites, since inhibitors, pointmutations in β subunits and the exchange of γ interferon-inducing βsubunits alter these activities to various degrees.

There are several examples of small molecules which have been used toinhibit proteasome activity; however, these compounds generally lack thespecificity, stability, or potency necessary to explore and exploit theroles of the proteasome at the cellular and molecular level. Therefore,the synthesis of small molecule inhibitor(s) with increased sitespecificity, improved stability and solubility, and increased potencyare needed to allow the exploration of the roles of the proteasome atthe cellular and molecular level.

SUMMARY OF THE INVENTION

The invention relates to classes of molecules known as peptideα′,β′-epoxides and peptide α′,β′-aziridines. The parent molecules areunderstood to bind efficiently, irreversibly and selectively toN-terminal nucleophile (Ntn) hydrolases, and can specifically inhibitparticular activities of enzymes having multiple catalytic activity.

Once thought merely to dispose of denatured and misfolded proteins, theproteasome is now recognized as constituting proteolytic machinery thatregulates the levels of diverse intracellular proteins through theirdegradation in a signal-dependent manner. Hence, there is great interestin identifying reagents that can specifically perturb the activities ofthe proteasome and other Ntn hydrolases and thereby be used as probes tostudy the role of these enzymes in biological processes. Compounds thattarget the NM hydrolases are herein described, synthesized, andinvestigated. Peptide epoxides and peptide aziridines that can potently,selectively, and irreversibly inhibit particular proteasome activitiesare disclosed and claimed.

Unlike several other peptide-based inhibitors, the peptide epoxides andpeptide aziridines described herein are not expected to substantiallyinhibit non-proteasomal proteases such as trypsin, chymotrypsin,cathepsin B, papain, and calpain at concentrations up to 50 μM. Athigher concentrations, inhibition may be observed, but would be expectedto be competitive and not irreversible, if the inhibitor merely competeswith the substrate. The novel peptide epoxides and peptide aziridinesare also expected to inhibit NF-κB activation and to stabilize p53levels in cell culture. Moreover, these compounds would be expected tohave anti-inflammatory activity. Thus, these compounds can be uniquemolecular probes, which have the versatility to explore Ntn enzymefunction in normal biological and pathological processes.

In one aspect, the invention provides inhibitors comprising aheteroatom-containing three-membered ring. These inhibitors can inhibitcatalytic activity of N-terminal nucleophile hydrolase enzymes (forexample, the 20S proteasome, or the 26S proteasome) when said inhibitoris present at concentrations below about 50 μM. Regarding the 20Sproteasome, particular hydrolase inhibitors inhibit chymotrypsin-likeactivity of the 20S proteasome when the inhibitor is present atconcentrations below about 5 μM, and does not inhibit trypsin-likeactivity or PGPH activity of the 20S proteasome when present atconcentrations below about 5 AM. The hydrolase inhibitor may be, forexample, a peptide α′,β′-epoxy ketone or α′,β′-aziridine ketone, and thepeptide may be a tetrapeptide. The peptide may include branched orunbranched side chains such as hydrogen, C₁₋₆alkyl, C₁₋₆hydroxyalkyl,C₁₋₆alkoxyalkyl, aryl, C₁₋₆aralkyl, C₁₋₆alkylamide, C₁₋₆alkylamine,C₁₋₆carboxylic acid, C₁₋₆carboxyl ester, C₁₋₆alkylthiol, or C₁₋₆alkylthioether, for example isobutyl, 1-naphthyl, phenylmethyl, and2-phenylethyl. The α′-carbon of the α′,β′-epoxy ketone orα′,β′-aziridine ketone may be a chiral carbon atom, such as an (R) or βconfigured carbon, as these are defined herein.

In another aspect, the invention provides pharmaceutical compositions,including a pharmaceutically acceptable carrier and a pharmaceuticallyeffective amount of the hydrolase inhibitor, which ameliorates theeffects of neurodegenerative disease (such as Alzheimer's disease),muscle-wasting diseases, cancer, chronic infectious diseases, fever,muscle disuse, denervation, nerve injury, fasting, and immune-relatedconditions, among others.

In another aspect, the invention provides anti-inflammatorycompositions.

In another aspect, the invention provides methods for the following:inhibiting or reducing HIV infection in a subject; affecting the levelof viral gene expression in a subject; altering the variety of antigenicpeptides produced by the proteasome in an organism; determining whethera cellular, developmental, or physiological process or output in anorganism is regulated by the proteolytic activity of a particular Ntnhydrolase; treating Alzheimer's disease in a subject; reducing the rateof muscle protein degradation in a cell; reducing the rate ofintracellular protein degradation in a cell; reducing the rate of p53protein degradation in a cell; inhibiting the growth of p53-relatedcancers in a subject; inhibiting antigen presentation in a cell;suppressing the immune system of a subject; inhibiting IκB-α degradationin an organism; reducing the content of NF-κB in a cell, muscle, organor subject; affecting cyclin-dependent eukaryotic cell cycles; treatingproliferative disease in a subject; affecting proteasome-dependentregulation of oncoproteins in a cell; treating cancer growth in asubject; treating p53-related apoptosis in a subject; and screeningproteins processed by N-terminal nucleophile hydrolases in a cell. Eachof these methods involves administering or contacting an effectiveamount of a composition comprising the hydrolase inhibitors disclosedherein, to a subject, a cell, a tissue, an organ, or an organism.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

The invention involves compounds useful as enzyme inhibitors. Thesecompounds are generally useful to inhibit enzymes having a nucleophilicgroup at the N-terminus. For example, activities of enzymes or enzymesubunits having N-terminal amino acids with nucleophiles in their sidechains, such as threonine, serine, or cysteine can be successfullyinhibited by the enzyme inhibitors described herein. Activities ofenzymes or enzyme subunits having non-amino acid nucleophilic groups attheir N-termini, such as, for example, protecting groups orcarbohydrates, can also be successfully inhibited by the enzymeinhibitors described herein.

While not bound by any particular theory of operation, it is believedthat such N-terminal nucleophiles of Ntn form covalent adducts with theepoxide functional group of the enzyme inhibitors described herein. Forexample, in the β5/Pre2 subunit of 20S proteasome, the N-terminalthreonine is believed to irreversibly form a morpholino or piperazinoadduct upon reaction with a peptide epoxide or aziridine such as thosedescribed below. Such adduct formation would involve ring-openingcleavage of the epoxide or aziridine.

In embodiments including such groups bonded to a′ carbons, thestereochemistry of the α′-carbon (that carbon forming a part of theepoxide or aziridine ring) can be (R) or (S). The invention is based, inpart, on the structure-function information disclosed herein, whichsuggests the following preferred stereochemical relationships. Note thata preferred compound may have a number of stereocenters having theindicated up-down (or β-α, where β as drawn herein is above the plane ofthe page) or (R)—(S) relationship (that is, it is not required thatevery stereocenter in the compound conform to the preferences stated).In some preferred embodiments, the stereochemistry of the α′ carbon is(R), that is, the X atom is β, or above the plane of the molecule.

Regarding the stereochemistry, the Cahn-Ingold-Prelog rules fordetermining absolute stereochemistry are followed. These rules aredescribed, for example, in Organic Chemistry, Fox and Whitesell; Jonesand Bartlett Publishers, Boston, Mass. (1994); Section 5-6, pp 177-178,which section is hereby incorporated by reference. Peptides can have arepeating backbone structure with side chains extending from thebackbone units. Generally, each backbone unit has a side chainassociated with it, although in some cases, the side chain is a hydrogenatom. In other embodiments, not every backbone unit has an associatedside chain. Peptides useful in peptide epoxides or peptide aziridineshave two or more backbone units. In some embodiments useful forinhibiting chymotrypsin-like (CT-L) activity of the proteasome, betweentwo and eight backbone units are present, and in some preferredembodiments for CT-L inhibition, between two and six backbone units arepresent.

The side chains extending from the backbone units can include naturalaliphatic or aromatic amino acid side chains, such as hydrogen(glycine), methyl (alanine), isopropyl (valine), sec-butyl (isoleucine),isobutyl (leucine), phenylmethyl (phenylalanine), and the side chainconstituting the amino acid proline. The side chains can also be otherbranched or unbranched aliphatic or aromatic groups such as ethyl,n-propyl, n-butyl, t-butyl, and aryl substituted derivatives such as1-phenylethyl, 2-phenylethyl, (1-naphthyl)methyl, (2-naphthyl)methyl,1-(1-naphthyl)ethyl, 1-(2-naphthyl)ethyl, 2-(1-naphthyl)ethyl,2-(2-naphthyl)ethyl, and similar compounds. The aryl groups can befurther substituted with branched or unbranched C₁₋₆alkyl groups, orsubstituted alkyl groups, acetyl and the like, or further aryl groups,or substituted aryl groups, such as benzoyl and the like. Heteroarylgroups can also be used as side chain substituents. Heteroaryl groupsinclude nitrogen-, oxygen-, and sulfur-containing aryl groups such asthienyl, benzothienyl, naphthothienyl, thianthrenyl, furyl, pyranyl,isobenzofuranyl, chromenyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl,pyrazinyl, indolyl, purinyl, quinolyl, and the like.

In some embodiments, polar or charged residues can be introduced intothe peptide epoxides or peptide aziridines. For example, naturallyoccurring amino acids such as hydroxy-containing (Thr, Tyr, Ser) orsulfur-containing (Met, Cys) can be introduced, as well as non-essentialamino acids, for example, taurine, carnitine, citrulline, cystine,ornithine, norleucine and others. Non-naturally occurring side chainsubstituents with charged or polar moieties can also be included, suchas, for example, C₁₋₆alkyl chains or C₆₋₁₂aryl groups with one or morehydroxy, short chain alkoxy, sulfide, thio, carboxyl, ester, phospho,amido or amino groups, or such substituents substituted with one or morehalogen atoms. In some preferred embodiments, there is at least one arylgroup present in a side chain of the peptide moiety.

In some embodiments, the backbone units are amide units[—NH—CHR—C(═O)—], in which R is the side chain. Such a designation doesnot exclude the naturally occurring amino acid proline, or othernon-naturally occurring cyclic secondary amino acids, which will berecognized by those of skill in the art.

In other embodiments, the backbone units are N-alkylated amide units(for example, N-methyl and the like), olefinic analogs (in which one ormore amide bonds are replaced by olefinic bonds), tetrazole analogs (inwhich a tetrazole ring imposes a cis-configuration on the backbone), orcombinations of such backbone linkages. In still other embodiments, theamino acid α-carbon is modified by α-alkyl substitution, for example,aminoisobutyric acid. In some further embodiments, side chains arelocally modified, for example, by ΔE or ΔZ dehydro modification, inwhich a double bond is present between the α and β atoms of the sidechain, or for example by ΔE or ΔZ cyclopropyl modification, in which acyclopropyl group is present between the α and β atoms of the sidechain. In still further embodiments employing amino acid groups, D-aminoacids can be used. Further embodiments can include sidechain-to-backbone cyclization, disulfide bond formation, lactamformation, azo linkage, and other modifications discussed in “Peptidesand Mimics, Design of Conformationally Constrained” by Hruby and Boteju,in “Molecular Biology and Biotechnology: A Comprehensive DeskReference”, ed. Robert A. Meyers, VCH Publishers (1995), pp. 658-664,which is hereby incorporated by reference.

In certain embodiments, the subject compounds have a structure offormula I or a pharmaceutically acceptable salt thereof,

wherein

L is absent or is selected from —CO₂ or —C(═S)O;

X is O, NH, or N-alkyl, preferably O;

Y is NH, N-alkyl, O, or C(R⁹)₂, preferably N-alkyl, O, or C(R⁹)₂;

Z is O or C(R⁹)₂, preferably C(R⁹)₂;

R¹, R², R³, and R⁴ are independently selected from hydrogen and a groupof formula II, preferably, R¹, R², R³, and R⁴ are all the same, morepreferably R¹, R², R³, and R⁴ are all hydrogen;

each R⁵, R⁶, R⁷, R⁸, and R⁹ is independently selected from hydrogen,C₁₋₆alkyl, C₁₋₆ hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl,each of which is optionally substituted with a group selected fromalkyl, amide, amine, carboxylic acid or a pharmaceutically acceptablesalt thereof, carboxyl ester, thiol, and thioether, preferably R⁵, R⁶,R⁷, and R⁸ are independently selected from C₁₋₆alkyl, C₁₋₆hydroxyalkyl,and C₁₋₆aralkyl and each R⁹ is hydrogen, more preferably, R⁶ and R⁸ areindependently C₁₋₆alkyl, R⁵ and R⁷ are independently C₁₋₆aralkyl andeach R⁹ is H;

R¹⁰ and R¹¹ are independently selected from hydrogen and C₁₋₆alkyl, orR¹⁰ and R¹¹ together form a 3- to 6-membered carbocyclic or heterocyclicring;

R¹² and R¹³ are independently selected from hydrogen, a metal cation,C₁₋₆alkyl, and C₁₋₆aralkyl, or R¹² and R¹³ together represent C₁₋₆alkyl,thereby forming a ring;

m is an integer from 0 to 2; and

n is an integer from 0 to 2, preferably 0 or 1.

In certain embodiments, X is O and R¹, R², R³, and R⁴ are all the same,preferably R¹, R², R³, and R⁴ are all hydrogen. In certain suchembodiments, R⁵, R⁶, R⁷, and R⁸ are independently selected fromC₁₋₆alkyl, C₁₋₆hydroxyalkyl, and C₁₋₆aralkyl, more preferably, R⁶ and R⁸are independently C₁₋₆alkyl and R⁵ and R⁷ are independently C₁₋₆aralkyl.

In certain preferred embodiments, X is O, R¹, R², R³, and R⁴ are allhydrogen, R⁶ and R⁸ are both isobutyl, R⁵ is phenylethyl, and R⁷ isphenylmethyl.

In certain embodiments, R⁵, R⁶, R⁷, and R⁸ are independently selectedfrom hydrogen, C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, andC₁₋₆aralkyl, each of which is optionally substituted with a groupselected from alkyl, amide, amine, carboxylic acid or a pharmaceuticallyacceptable salt thereof, carboxyl ester, thiol, and thioether. Incertain embodiments, at least one of R⁵ and R⁷ is C₁₋₆aralkylsubstituted with alkyl, more preferably substituted with perhaloalkyl.In certain such embodiments, R⁷ is C₁₋₆aralkyl substituted withtrifluoromethyl.

In certain embodiments, Y is selected from N-alkyl, O, and CH₂. Incertain such embodiments, Z is CH₂, and m and n are both 0. In certainalternative such embodiments, Z is CH₂, m is 0, and n is 2 or 3. In yetanother alternative such embodiments, Z is O, m is 1, and n is 2.

In certain embodiments, a compound of formula I is selected from

In certain embodiments, the subject compounds have a structure offormula III or a pharmaceutically acceptable salt thereof,

where X is O, NH, or N-alkyl, preferably O;

R¹, R², R³, and R⁴ are independently selected from hydrogen and a groupof formula II, preferably, R¹, R², R³, and R⁴ are all the same, morepreferably R¹, R², R³, and R⁴ are all hydrogen; and

R⁵, R⁶, R⁷, and R⁸ are independently selected from hydrogen, C₁₋₆alkyl,C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, each of whichis optionally substituted with a group selected from amide, amine,carboxylic acid or a pharmaceutically acceptable salt thereof, carboxylester, thiol, and thioether, preferably R⁵, R⁶, R⁷, and R⁸ areindependently selected from C₁₋₆alkyl, C₁ hydroxyalkyl, and C₁₋₆aralkyl,more preferably, R⁶ and R⁸ are independently C₁₋₆alkyl and R⁵ and R⁷ areindependently C₁₋₆aralkyl.

In certain embodiments, X is O and R¹, R², R³, and R⁴ are all the same,preferably R¹, R², R³, and R⁴ are all hydrogen. In certain suchembodiments, R⁵, R⁶, R⁷, and R⁸ are independently selected fromC₁₋₆alkyl, C₁₋₆hydroxyalkyl, and C₁₋₆aralkyl, more preferably, R⁶ and R⁸are independently C₁₋₆alkyl and R⁵ and R⁷ are independently C₁₋₆aralkyl.

In certain preferred embodiments, X is O, R¹, R², R³, and R⁴ are allhydrogen, R⁶ and R⁸ are both isobutyl, R⁵ is phenylethyl, and R⁷ isphenylmethyl.

In certain embodiments, a compound of formula III has the followingstereochemistry:

In preferred embodiments, the compound has a structure of formula IV ora pharmaceutically acceptable salt thereof,

wherein

X is O, NH, or N-alkyl, preferably O;

R¹, R², R³, and R⁴ are independently selected from hydrogen and a groupof formula II, preferably, R¹, R², R³, and R⁴ are all the same, morepreferably R¹, R², R³, and R⁴ are all hydrogen; and

R⁶ and R⁸ are independently selected from hydrogen, C₁₋₆alkyl,C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, each of whichis optionally substituted with a group selected from amide, amine,carboxylic acid or a pharmaceutically acceptable salt thereof, carboxylester, thiol, and thioether, preferably R⁶ and R⁸ are independentlyselected from C₁₋₆alkyl, C₁₋₆hydroxyalkyl, and C₁₋₆aralkyl, morepreferably, R⁶ and R⁸ are independently C₁₋₆alkyl.

In certain embodiments, X is O and R¹, R², R³, and R⁴ are all the same,preferably R¹, R², R³, and R⁴ are all hydrogen. In certain suchembodiments, R⁶ and R⁸ are independently selected from C₁₋₆alkyl,C₁₋₆hydroxyalkyl, and C₁₋₆aralkyl, more preferably, R⁶ and R⁸ areindependently C₁₋₆alkyl.

In certain preferred embodiments, X is O, R¹, R², R³, and R⁴ are allhydrogen, and R⁶ and R⁸ are both isobutyl.

In certain embodiments, a compound of formula III has the followingstructure:

In certain embodiments, the compounds have a structure of formula V or apharmaceutically acceptable salt thereof

wherein

X is O, NH, or N-alkyl, preferably O;

R¹, R², R³, and R⁴ are independently selected from hydrogen and a groupof formula II, preferably, R¹, R², R³, and R⁴ are all the same, morepreferably R¹, R², R³, and R⁴ are all hydrogen;

R⁵, R⁶, R⁷, and R⁸ are independently selected from hydrogen, C₁₋₆alkyl,C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, each of whichis optionally substituted with a group selected from amide, amine,carboxylic acid or a pharmaceutically acceptable salt thereof, carboxylester, thiol, and thioether, preferably R⁵, R⁶, R⁷, and R⁸ areindependently selected from C₁₋₆ alkyl, C₁₋₆ hydroxyalkyl, andC₁₋₆aralkyl, more preferably, R⁶ and R⁸ are independently C₁₋₆alkyl andR⁵ and R⁷ are independently C₁. 6aralkyl; and

q is an integer from 0 to 3.

In preferred embodiments, the compound has a structure of formula VI ora pharmaceutically acceptable salt thereof,

wherein

X is O, NH, or N-alkyl, preferably O;

R¹, R², R³, and R⁴ are independently selected from hydrogen and a groupof formula II, preferably, R¹, R², R³, and R⁴ are all the same, morepreferably R¹, R², R³, and R⁴ are all hydrogen;

R⁶ and R⁸ are independently selected from hydrogen, C₁₋₆alkyl,C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, each of whichis optionally substituted with a group selected from amide, amine,carboxylic acid or a pharmaceutically acceptable salt thereof, carboxylester, thiol, and thioether, preferably R⁶ and R⁸ are independentlyselected from C₁₋₆alkyl, C₁₋₆hydroxyalkyl, and C₁₋₆aralkyl, morepreferably, R⁶ and R⁸ are independently C₁₋₆ alkyl; and

q is an integer from 0 to 3.

In certain embodiments, X is O and R¹, R², R³, and R⁴ are all the same,preferably R¹, R², R³, and R⁴ are all hydrogen. In certain suchembodiments, R⁶ and R⁸ are independently selected from C₁₋₆alkyl,C₁₋₆hydroxyalkyl, and C₁₋₆aralkyl, more preferably, R⁶ and R⁸ areindependently C₁₋₆alkyl.

In certain preferred embodiments, X is O, R¹, R², R³, and R⁴ are allhydrogen, and R⁶ and R⁸ are both isobutyl.

In certain embodiments, a compound of formula VI is selected from

One aspect of the invention relates to a medical device includingcomposition disclosed herein that include an inhibitor having astructure of any one of formulae I or III-VI. In one embodiment, thecomposition is incorporated within a medical device. In certainembodiments, the medical device is a gel comprising a polymer matrix orceramic matrix and an inhibitor. Said polymer can be either naturallyoccurring or synthetic. In another embodiment, said gel serves as a drugdepot, an adhesive, a suture, a barrier or a sealant.

Another aspect of the invention relates to a medical device comprising asubstrate having a surface onto which an inhibitor having a structure ofany one of formulae I or III-VI is disposed. In one embodiment, theinhibitor is directly disposed on a medical device. In anotherembodiment, a coating is so disposed, the coating comprising a polymermatrix or ceramic matrix with an inhibitor having a structure of any oneof formulae I or III-VI dispersed or dissolved therein.

In one embodiment, the medical device is a coronary, vascular,peripheral, or biliary stent. More particularly, the stent of thepresent invention is an expandable stent. When coated with a matrixcontaining an inhibitor having a structure of any one of formulae I orthe matrix is flexible to accommodate compressed and expanded states ofsuch an expandable stent. In another embodiment of this invention, thestent has at least a portion which is insertable or implantable into thebody of a patient, wherein the portion has a surface which is adaptedfor exposure to body tissue and wherein at least a part of the surfaceis coated with an inhibitor having a structure of any one of formulae Ior or a coating comprising a matrix having an inhibitor having astructure of any one of formulae I or III-VI is dispersed or dissolvedtherein. An example of a suitable stent is disclosed in U.S. Pat. No.4,733,665, which is incorporated herein by reference in its entirety.

In another embodiment, the medical device of the present invention is asurgical implement such as a vascular implant, an intraluminal device,surgical sealant or a vascular support. More particularly, the medicaldevice of the present invention is a catheter, an implantable vascularaccess port, a central venous catheter, an arterial catheter, a vasculargraft, an intraaortic balloon pump, a suture, a ventricular assist pump,a drug-eluting barrier, an adhesive, a vascular wrap, anextra/perivascular support, a blood filter, or a filter adapted fordeployment in a blood vessel, coated with an inhibitor having astructure of any one of formulae I or III-VI either directly or by amatrix containing an inhibitor having a structure of any one of formulaeI or

In certain embodiments, the intraluminal medical device is coated withan inhibitor having a structure of any one of formulae I or III-VI or acoating comprising biologically tolerated matrix and an inhibitor havinga structure of any one of formulae I or III-VI dispersed in the polymer,said device having an interior surface and an exterior surface, havingthe coating applied to at least a part of the interior surface, theexterior surface, or both.

In certain embodiments, the medical device may be useful to preventrestenosis after angioplasty. The medical device may also be useful forthe treatment of various diseases and conditions by providing localizedadministration of an inhibitor having a structure of any one of formulaeI or Such diseases and conditions include restenosis, inflammation,rheumatoid arthritis, tissue injury due to inflammation,hyperproliferative diseases, severe or arthritic psoriasis,muscle-wasting diseases, chronic infectious diseases, abnormal immuneresponse, conditions involving vulnerable plaques, injuries related toischemic conditions, and viral infection and proliferation. Examples ofdiseases and conditions that are subject to a treatment including thedrug coated medical devices of the present invention includeatherosclerosis, acute coronary syndrome, Alzheimer's disease, cancer,fever, muscle disuse (atrophy), denervation, vascular occlusions,stroke, HIV infection, nerve injury, renal failure associated withacidosis, and hepatic failure. See, e.g., Goldberg, U.S. Pat. No.5,340,736.

The term “C_(x-y)alkyl” refers to substituted or unsubstituted saturatedhydrocarbon groups, including straight-chain alkyl and branched-chainalkyl groups that contain from x to y carbons in the chain, includinghaloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc.C₀alkyl indicates a hydrogen where the group is in a terminal position,a bond if internal. The terms “C_(2-y)alkenyl” and “C_(2-y)alkynyl”refer to substituted or unsubstituted unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The term “alkoxy” refers to an alkyl group having an oxygen attachedthereto. Representative alkoxy groups include methoxy, ethoxy, propoxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxy.

The term “C₁₋₆alkoxyalkyl” refers to a C₁₋₆alkyl group substituted withan alkoxy group, thereby forming an ether.

The term “C₁₋₆aralkyl”, as used herein, refers to a C₁₋₆alkyl groupsubstituted with an aryl group.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by the general formulae:

wherein R⁹, R¹⁰ and R^(10′) each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R⁸, or R⁹ and R¹⁰ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R⁸ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocyclyl or a polycyclyl; and m is zero or aninteger from 1 to 8. In preferred embodiments, only one of R⁹ or R¹⁰ canbe a carbonyl, e.g., R⁹, R¹⁰, and the nitrogen together do not form animide. In even more preferred embodiments, R⁹ and R¹⁰ (and optionallyR^(10′)) each independently represent a hydrogen, an alkyl, an alkenyl,or —(CH₂)_(m)—R⁸. In certain embodiments, the amino group is basic,meaning the protonated form has a pK_(a)≧7.00.

The terms “amide” and “amido” are art-recognized as an amino-substitutedcarbonyl and includes a moiety that can be represented by the generalformula:

wherein R⁹, R¹⁰ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “aryl” as used herein includes 5-, 6-, and 7-memberedsubstituted or unsubstituted single-ring aromatic groups in which eachatom of the ring is carbon. The term “aryl” also includes polycyclicring systems having two or more cyclic rings in which two or morecarbons are common to two adjoining rings wherein at least one of therings is aromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline,and the like.

The terms “carbocycle” and “carbocyclyl”, as used herein, refer to anon-aromatic substituted or unsubstituted ring in which each atom of thering is carbon. The terms “carbocycle” and “carbocyclyl” also includepolycyclic ring systems having two or more cyclic rings in which two ormore carbons are common to two adjoining rings wherein at least one ofthe rings is carbocyclic, e.g., the other cyclic rings can becycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R¹¹represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R⁸ or apharmaceutically acceptable salt, R¹ represents a hydrogen, an alkyl, analkenyl or —(CH₂)_(m)—R⁸, where m and R⁸ are as defined above. Where Xis an oxygen and R¹¹ or R^(11′) is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R¹¹ is a hydrogen, the formularepresents a “carboxylic acid”.

As used herein, “enzyme” can be any partially or wholly proteinaceousmolecule which carries out a chemical reaction in a catalytic manner.Such enzymes can be native enzymes, fusion enzymes, proenzymes,apoenzymes, denatured enzymes, farnesylated enzymes, ubiquitinatedenzymes, fatty acylated enzymes, gerangeranylated enzymes, GPI-linkedenzymes, lipid-linked enzymes, prenylated enzymes, naturally-occurringor artificially-generated mutant enzymes, enzymes with side chain orbackbone modifications, enzymes having leader sequences, and enzymescomplexed with non-proteinaceous material, such as proteoglycans,proteoliposomes. Enzymes can be made by any means, including naturalexpression, promoted expression, cloning, various solution-based andsolid-based peptide syntheses, and similar methods known to those ofskill in the art.

The term “C₁₋₆heteroaralkyl”, as used herein, refers to a C₁₋₆alkylgroup substituted with a heteroaryl group.

The terms “heteroaryl” includes substituted or unsubstituted aromatic 5-to 7-membered ring structures, more preferably 5- to 6-membered rings,whose ring structures include one to four heteroatoms. The term“heteroaryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings wherein at least one of the rings is heteroaromatic, e.g., theother cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, forexample, pyrrole, furan, thiophene, imidazole, oxazole, thiazole,triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, andthe like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,phosphorus, and sulfur.

The terms “heterocyclyl” or “heterocyclic group” refer to substituted orunsubstituted non-aromatic 3- to 10-membered ring structures, morepreferably 3- to 7-membered rings, whose ring structures include one tofour heteroatoms. The term terms “heterocyclyl” or “heterocyclic group”also include polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings wherein atleast one of the rings is heterocyclic, e.g., the other cyclic rings canbe cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls. Heterocyclyl groups include, for example, piperidine,piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

The term “C₁₋₆hydroxyalkyl” refers to a C₁₋₆alkyl group substituted witha hydroxy group.

As used herein, the term “inhibitor” is meant to describe a compoundthat blocks or reduces an activity of an enzyme (for example, inhibitionof proteolytic cleavage of standard fluorogenic peptide substrates suchas suc-LLVY-AMC, Box-LLR-AMC and Z-LLE-AMC, inhibition of variouscatalytic activities of the 20S proteasome). An inhibitor can act withcompetitive, uncompetitive, or noncompetitive inhibition. An inhibitorcan bind reversibly or irreversibly, and therefore the term includescompounds that are suicide substrates of an enzyme. An inhibitor canmodify one or more sites on or near the active site of the enzyme, or itcan cause a conformational change elsewhere on the enzyme.

As used herein, the term “peptide” includes not only standard amidelinkage with standard α-substituents, but commonly utilizedpeptidomimetics, other modified linkages, non-naturally occurring sidechains, and side chain modifications, as detailed below.

The terms “polycyclyl” or “polycyclic” refer to two or more rings (e.g.,cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Each of the rings of thepolycycle can be substituted or unsubstituted.

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive the composition. Thus, prevention of cancer includes,for example, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount. Prevention of an infection includes, for example,reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

The term “prodrug” encompasses compounds that, under physiologicalconditions, are converted into therapeutically active agents. A commonmethod for making a prodrug is to include selected moieties that arehydrolyzed under physiological conditions to reveal the desiredmolecule. In other embodiments, the prodrug is converted by an enzymaticactivity of the host animal.

The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The term “proteasome” as used herein is meant to include immuno- andconstitutive proteasomes.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include, for example, a halogen, ahydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl,or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or athioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, aphosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro,an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, asulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or anaromatic or heteroaromatic moiety. It will be understood by thoseskilled in the art that the moieties substituted on the hydrocarbonchain can themselves be substituted, if appropriate.

A “therapeutically effective amount” of a compound with respect to thesubject method of treatment, refers to an amount of the compound(s) in apreparation which, when administered as part of a desired dosage regimen(to a mammal, preferably a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to be,treated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment.

The term “thioether” refers to an alkyl group, as defined above, havinga sulfur moiety attached thereto. In preferred embodiments, the“thioether” is represented by —S— alkyl. Representative thioether groupsinclude methylthio, ethylthio, and the like.

As used herein, the term “treating” or “treatment” includes reversing,reducing, or arresting the symptoms, clinical signs, and underlyingpathology of a condition in manner to improve or stabilize a subject'scondition.

Selectivity for 20S Proteasome

The enzyme inhibitors disclosed herein are useful in part because theyinhibit the action of the 20S proteasome. Additionally, unlike other 20Sproteasome inhibitors, the compounds disclosed herein are highlyselective toward the 20S proteasome, with respect to other proteaseenzymes. That is, the instant compounds show selectivities for the 20Sproteasome over other proteases such as cathepsins, calpains, papain,chymotrypsin, trypsin, tripeptidyl pepsidase II. The selectivities ofthe enzyme inhibitors for 20S proteasome are such that at concentrationsbelow about 50 μM, the enzyme inhibitors show inhibition of thecatalytic activity of the 20S proteasome, while not showing inhibitionof the catalytic activity of other proteases such as cathepsins,calpains, papain, chymotrypsin, trypsin, tripeptidyl pepsidase II. Inpreferred embodiments, the enzyme inhibitors show inhibition of thecatalytic activity of the 20S proteasome at concentrations below about10 μM, while not showing inhibition of the catalytic activity of otherproteases at these concentrations. In even more preferred embodiments,the enzyme inhibitors show inhibition of the catalytic activity of the20S proteasome at concentrations below about 1 μM, while not showinginhibition of the catalytic activity of other proteases at theseconcentrations. Enzyme kinetic assays are disclosed in U.S. applicationSer. No. 09/569,748, Example 2 and Stein et al., Biochem. (1996), 35,3899-3908.

Selectivity for Chymotrypsin-Like Activity

Particular embodiments of the enzyme inhibiting compounds describedherein are further useful because they can efficiently and selectivelyinhibit the chymotrypsin-like activity of the 20S proteasome, ascompared to the trypsin-like, and PGPH activities. The chymotrypsin-likeactivity of 20S proteasome is characterized by cleavage of peptides inthe immediate vicinity of large hydrophobic residues. In particular, thechymotrypsin-like activity of Ntn hydrolases can be determined bycleavage of a standard substrate. Examples of such substrates are knownin the art. For example, a leucylvalinyltyrosine derivative can be used.Enzyme kinetic assays are disclosed in U.S. application Ser. No.09/569,748, Example 2 and Stein et al., Biochem. (1996), 35, 3899-3908.

Uses of Enzyme Inhibitors

The biological consequences of proteasome inhibition are numerous. Atthe cellular level, the accumulation of polyubiquitinated proteins, cellmorphological changes, and apoptosis have been reported upon treatmentof cells with various proteasome inhibitors. Proteasome inhibition hasalso been suggested as a possible antitumor therapeutic strategy. Thefact that epoxomicin was initially identified in a screen for antitumorcompounds validates the proteasome as an antitumor chemotherapeutictarget. Accordingly, these compounds are useful for treating cancer.Proteasome inhibition has also been associated with inhibition of NF-κBactivation and stabilization of p53 levels. Thus, compounds of theinvention may also be used to inhibit NF-κB activation, and stabilizep53 levels in cell culture. Since NF-κB is a key regulator ofinflammation, it is an attractive target for anti-inflammatorytherapeutic intervention. Thus, compounds of the invention may be usefulfor the treatment of conditions associated with chronic inflammation,including, but not limited to COPD, psoriasis, bronchitis, emphysema,and cystic fibrosis.

The disclosed compounds can be used to treat conditions mediateddirectly by the proteolytic function of the proteasome such as musclewasting, or mediated indirectly via proteins which are processed by theproteasome such as NF-κB. The proteasome participates in the rapidelimination and post-translational processing of proteins (e.g.,enzymes) involved in cellular regulation (e.g., cell cycle, genetranscription, and metabolic pathways), intercellular communication, andthe immune response (e.g., antigen presentation). Specific examplesdiscussed below include β-amyloid protein and regulatory proteins suchas cyclins, TGF-β, and transcription factor NF-κB.

Another embodiment of the invention is the use of the compoundsdisclosed herein for the treatment of neurodegenerative diseases andconditions, including, but not limited to, stroke, ischemic damage tothe nervous system, neural trauma (e.g., percussive brain damage, spinalcord injury, and traumatic damage to the nervous system), multiplesclerosis and other immune-mediated neuropathies (e.g., Guillain-Barresyndrome and its variants, acute motor axonal neuropathy, acuteinflammatory demyelinating polyneuropathy, and Fisher Syndrome),HIV/AIDS dementia complex, axonomy, diabetic neuropathy, Parkinson'sdisease, Huntington's disease, multiple sclerosis, bacterial, parasitic,fungal, and viral meningitis, encephalitis, vascular dementia,multi-infarct dementia, Lewy body dementia, frontal lobe dementia suchas Pick's disease, subcortical dementias (such as Huntington orprogressive supranuclear palsy), focal cortical atrophy syndromes (suchas primary aphasia), metabolic-toxic dementias (such as chronichypothyroidism or B12 deficiency), and dementias caused by infections(such as syphilis or chronic meningitis).

Alzheimer's disease is characterized by extracellular deposits ofβ-amyloid protein (β-AP) in senile plaques and cerebral vessels. β-AP isa peptide fragment of 39 to 42 amino acids derived from an amyloidprotein precursor (APP). At least three isoforms of APP are known (695,751, and 770 amino acids). Alternative splicing of mRNA generates theisoforms; normal processing affects a portion of the β-AP sequence,thereby preventing the generation of β-AP. It is believed that abnormalprotein processing by the proteasome contributes to the abundance ofβ-AP in the Alzheimer brain. The APP-processing enzyme in rats containsabout ten different subunits (22 kDa-32 kDa). The 25 kDa subunit has anN-terminal sequence of X-Gln-Asn-Pro-Met-X-Thr-Gly-Thr-Ser, which isidentical to the β-subunit of human macropain (Kojima, S. et al., Fed.Eur. Biochem. Soc., (1992) 304:57-60). The APP-processing enzyme cleavesat the Gln¹⁵-Lys¹⁶ bond; in the presence of calcium ion, the enzyme alsocleaves at the Met-¹-Asp¹ bond, and the Asp¹-Ala² bonds to release theextracellular domain of 3-AP.

One embodiment, therefore, is a method of treating Alzheimer's disease,including administering to a subject an effective amount of a compound(e.g., pharmaceutical composition) disclosed herein. Such treatmentincludes reducing the rate of β-AP processing, reducing the rate of β-APplaque formation, reducing the rate of β-AP generation, and reducing theclinical signs of Alzheimer's disease.

Other embodiments of the invention relate to cachexia and muscle-wastingdiseases. The proteasome degrades many proteins in maturingreticulocytes and growing fibroblasts. In cells deprived of insulin orserum, the rate of proteolysis nearly doubles. Inhibiting the proteasomereduces proteolysis, thereby reducing both muscle protein loss and thenitrogenous load on kidneys or liver. Inhibitors of the invention areuseful for treating conditions such as cancer, chronic infectiousdiseases, fever, muscle disuse (atrophy) and denervation, nerve injury,fasting, renal failure associated with acidosis, diabetes, and hepaticfailure. See, e.g., Goldberg, U.S. Pat. No. 5,340,736. Embodiments ofthe invention therefore encompass methods for: reducing the rate ofmuscle protein degradation in a cell; reducing the rate of intracellularprotein degradation; reducing the rate of degradation of p53 protein ina cell; and inhibiting the growth of p53-related cancers. Each of thesemethods includes contacting a cell (in vivo or in vitro, e.g., a musclein a subject) with an effective amount of a compound (e.g.,pharmaceutical composition) disclosed herein.

Fibrosis is the excessive and persistent formation of scar tissueresulting from the hyperproliferative growth of fibroblasts and isassociated with activation of the TGF-β signaling pathway. Fibrosisinvolves extensive deposition of extracellular matrix and can occurwithin virtually any tissue or across several different tissues.Normally, the level of intracellular signaling protein (Smad) thatactivate transcription of target genes upon TGF-β stimulation isregulated by proteasome activity (Xu et al., 2000). However, accelerateddegradation of the TGF-β signaling components has been observed incancers and other hyperproliferative conditions. Thus, certainembodiments of the invention relate to a method for treatinghyperproliferative conditions such as diabetic retinopathy, maculardegeneration, diabetic nephropathy, glomerulosclerosis, IgA nephropathy,cirrhosis, biliary atresia, congestive heart failure, scleroderma,radiation-induced fibrosis, and lung fibrosis (idiopathic pulmonaryfibrosis, collagen vascular disease, sarcoidosis, interstial lungdiseases and extrinsic lung disorders). The treatment of burn victims isoften hampered by fibrosis, thus, an additional embodiment of theinvention is the topical or systemic administration of the inhibitors totreat burns. Wound closure following surgery is often associated withdisfiguring scars, which may be prevented by inhibition of fibrosis.Thus, in certain embodiments, the invention relates to a method for theprevention or reduction of scarring.

Another protein processed by the proteasome is NF-κB, a member of theRel protein family. The Rel family of transcriptional activator proteinscan be divided into two groups. The first group requires proteolyticprocessing, and includes p50 (NF-κB1, 105 kDa) and p52 (NF-κ2, 100 kDa).The second group does not require proteolytic processing, and includesp65 (RelA, Rel (c-Rel), and RelB). Both homo- and heterodimers can beformed by Rel family members; NF-κB, for example, is a p50-p65heterodimer. After phosphorylation and ubiquitination of IκB and p105,the two proteins are degraded and processed, respectively, to produceactive NF-κB which translocates from the cytoplasm to the nucleus.Ubiquitinated p105 is also processed by purified proteasomes (Palombellaet al., Cell (1994) 78:773-785). Active NF-κB forms a stereospecificenhancer complex with other transcriptional activators and, e.g., HMGI(Y), inducing selective expression of a particular gene.

NF-κB regulates genes involved in the immune and inflammatory response,and mitotic events. For example, NF-κB is required for the expression ofthe immunoglobulin light chain K gene, the IL-2 receptor α-chain gene,the class I major histocompatibility complex gene, and a number ofcytokine genes encoding, for example, IL-2, IL-6, granulocytecolony-stimulating factor, and IFN-β (Palombella et al., Cell (1994)78:773-785). Some embodiments of the invention include methods ofaffecting the level of expression of IL-2, MHC-I, IL-6, TNFα, IFN-β orany of the other previously-mentioned proteins, each method includingadministering to a subject an effective amount of a compound disclosedherein. Complexes including p50 are rapid mediators of acuteinflammatory and immune responses (Thanos, D. and Maniatis, T., Cell(1995) 80:529-532).

NF-κB also participates in the expression of the cell adhesion genesthat encode E-selectin, P-selectin, ICAM, and VCAM-1 (Collins, T., Lab.Invest. (1993) 68:499-508). One embodiment of the invention is a methodfor inhibiting cell adhesion (e.g., cell adhesion mediated byE-selectin, P-selectin, ICAM, or VCAM-1), including contacting a cellwith (or administering to a subject) an effective amount of a compound(or a pharmaceutical composition) disclosed herein.

Ischemia and reperfusion injury results in hypoxia, a condition in whichthere is a deficiency of oxygen reaching the tissues of the body. Thiscondition causes increased degradation of Iκ-Bα, thereby resulting inthe activation of NF-κB (Koong et al., 1994). It has been demonstratedthat the severity of injury resulting in hypoxia can be reduced with theadministration of a proteasome inhibitor (Gao et al., 2000; Bao et al.,2001; Pye et al., 2003). Therefore, certain embodiments of the inventionrelate to a method of treating an ischemic condition or reperfusioninjury comprising administering to a subject in need of such treatmentan effective amount of a compound disclosed herein. Examples of suchconditions or injuries include, but are not limited to, acute coronarysyndrome (vulnerable plaques), arterial occlusive disease (cardiac,cerebral, peripheral arterial and vascular occlusions), atherosclerosis(coronary sclerosis, coronary artery disease), infarctions, heartfailure, pancreatitis, myocardial hypertrophy, stenosis, and restenosis.

NF-κB also binds specifically to the HIV-enhancer/promoter. Whencompared to the Nef of mac239, the HIV regulatory protein Nef of pbj14differs by two amino acids in the region which controls protein kinasebinding. It is believed that the protein kinase signals thephosphorylation of IκB, triggering IκB degradation through theubiquitin-proteasome pathway. After degradation, NF-κB is released intothe nucleus, thus enhancing the transcription of HIV (Cohen, J.,Science, (1995) 267:960). Two embodiments of the invention are a methodfor inhibiting or reducing HIV infection in a subject, and a method fordecreasing the level of viral gene expression, each method includingadministering to the subject an effective amount of a compound disclosedherein.

Overproduction of lipopolysaccharide (LPS)-induced cytokines such asTNFα is considered to be central to the processes associated with septicshock. Furthermore, it is generally accepted that the first step in theactivation of cells by LPS is the binding of LPS to specific membranereceptors. The α- and β-subunits of the 20S proteasome complex have beenidentified as LPS-binding proteins, suggesting that the LPS-inducedsignal transduction may be an important therapeutic target in thetreatment or prevention of sepsis (Qureshi, N. et al., J. Immun. (2003)171: 1515-1525). Therefore, in certain embodiments, compounds of theinvention may be used for the inhibition of TNFα to prevent and/or treatseptic shock.

Intracellular proteolysis generates small peptides for presentation toT-lymphocytes to induce MHC class I-mediated immune responses. Theimmune system screens for autologous cells that are virally infected orhave undergone oncogenic transformation. One embodiment is a method forinhibiting antigen presentation in a cell, including exposing the cellto a compound described herein. A compound of the invention may be usedto treat immune-related conditions such as allergy, asthma, organ/tissuerejection (graft-versus-host disease), and auto-immune diseases,including, but not limited to, lupus, rheumatoid arthritis, psoriasis,multiple sclerosis, and inflammatory bowel diseases (such as ulcerativecolitis and Crohn's disease). Thus, a further embodiment is a method forsuppressing the immune system of a subject (e.g., inhibiting transplantrejection, allergies, auto-immune diseases, and asthma), includingadministering to the subject an effective amount of a compound describedherein.

Another further embodiment is a method for altering the repertoire ofantigenic peptides produced by the proteasome or other Ntn withmulticatalytic activity. For example, if the PGPH activity of 20Sproteasome is selectively inhibited, a different set of antigenicpeptides will be produced by the proteasome and presented in MHCmolecules on the surfaces of cells than would be produced and presentedeither without any enzyme inhibition, or with, for example, selectiveinhibition of chymotrypsin-like activity of the proteasome.

Certain proteasome inhibitors block both degradation and processing ofubiquitinated NF-κB in vitro and in vivo. Proteasome inhibitors alsoblock IκB-α degradation and NF-κB activation (Palombella, et al. Cell(1994) 78:773-785; and Traenckner, et al., EMBO J. (1994) 13:5433-5441).One embodiment of the invention is a method for inhibiting IκB-αdegradation, including contacting the cell with a compound describedherein. A further embodiment is a method for reducing the cellularcontent of NF-κB in a cell, muscle, organ, or subject, includingcontacting the cell, muscle, organ, or subject with a compound describedherein.

Other eukaryotic transcription factors that require proteolyticprocessing include the general transcription factor TFIIA, herpessimplex virus VP16 accessory protein (host cell factor), virus-inducibleIFN regulatory factor 2 protein, and the membrane-bound sterolregulatory element-binding protein 1.

Other embodiments of the invention are methods for affectingcyclin-dependent eukaryotic cell cycles, including exposing a cell (invitro or in vivo) to a compound disclosed herein. Cyclins are proteinsinvolved in cell cycle control. The proteasome participates in thedegradation of cyclins. Examples of cyclins include mitotic cyclins, G1cyclins, and cyclin B. Degradation of cyclins enables a cell to exit onecell cycle stage (e.g., mitosis) and enter another (e.g., division). Itis believed all cyclins are associated with p34.sup.cdc2 protein kinaseor related kinases. The proteolysis targeting signal is localized toamino acids 42-RAALGNISEN-50 (destruction box). There is evidence thatcyclin is converted to a form vulnerable to a ubiquitin ligase or that acyclin-specific ligase is activated during mitosis (Ciechanover, A.,Cell, (1994) 79:13-21). Inhibition of the proteasome inhibits cyclindegradation, and therefore inhibits cell proliferation, for example, incyclin-related cancers (Kumatori et al., Proc. Natl. Acad. Sci. USA(1990) 87:7071-7075). One embodiment of the invention is a method fortreating a proliferative disease in a subject (e.g., cancer, psoriasis,or restenosis), including administering to the subject an effectiveamount of a compound disclosed herein. The invention also encompasses amethod for treating cyclin-related inflammation in a subject, includingadministering to a subject a therapeutically effective amount of acompound described herein.

Additional embodiments are methods for affecting theproteasome-dependent regulation of oncoproteins and methods of treatingor inhibiting cancer growth, each method including exposing a cell (invivo, e.g., in a subject, or in vitro) to a compound disclosed herein.HPV-16 and HPV-18-derived E6 proteins stimulate ATP- andubiquitin-dependent conjugation and degradation of p53 in crudereticulocyte lysates. The recessive oncogene p53 has been shown toaccumulate at the nonpermissive temperature in a cell line with amutated thermolabile E1. Elevated levels of p53 may lead to apoptosis.Examples of proto-oncoproteins degraded by the ubiquitin system includec-Mos, c-Fos, and c-Jun. One embodiment is a method for treatingp53-related apoptosis, including administering to a subject an effectiveamount of a compound disclosed herein.

In another embodiment, the disclosed compounds are useful for thetreatment of a parasitic infection, such as infections caused byprotozoan parasites. The proteasome of these parasites is considered tobe involved primarily in cell differentiation and replication activities(Paugam et al., Trends Parasitol. 2003, 19(2): 55-59). Furthermore,entamoeba species have been shown to lose encystation capacity whenexposed to proteasome inhibitors (Gonzales, et al., Arch. Med. Res.1997, 28, Spec No: 139-140). In certain such embodiments, the disclosedcompounds are useful for the treatment of parasitic infections in humanscaused by a protozoan parasite selected from Plasmodium sps. (includingP. falciparum, P. vivax, P. malariae, and P. ovale, which causemalaria), Trypanosoma sps. (including T. cruzi, which causes Chagas'disease, and T. brucei which causes African sleeping sickness),Leishmania sps. (including L. amazonensis, L. donovani, L. infantum, L.mexicana, etc.), Pneumocystis carinii (a protozoan known to causepneumonia in AIDS and other immunosuppressed patients), Toxoplasmagondii, Entamoeba histolytica, Entamoeba invadens, and Giardia lamblia.In certain embodiments, the disclosed compounds are useful for thetreatment of parasitic infections in animals and livestock caused by aprotozoan parasite selected from Plasmodium hermani, Cryptosporidiumsps., Echinococcus granulosus, Eimeria tenella, Sarcocystis neurona, andNeurospora crassa. Other compounds useful as proteasome inhibitors inthe treatment of parasitic diseases are described in WO 98/10779, whichis incorporated herein in its entirety.

In certain embodiments, the disclosed compounds inhibit proteasomeactivity irreversibly in a parasite. Such irreversible inhibition hasbeen shown to induce shutdown in enzyme activity without recovery in redblood cells and white blood cells. In certain such embodiments, the longhalf-life of blood cells may provide prolonged protection with regard totherapy against recurring exposures to parasites. In certainembodiments, the long half-life of blood cells may provide prolongedprotection with regard to chemoprophylaxis against future infection.

It has also been demonstrated that inhibitors that bind to the 20Sproteasome stimulate bone formation in bone organ cultures. Furthermore,when such inhibitors have been administered systemically to mice,certain proteasome inhibitors increased bone volume and bone formationrates over 70% (Garrett, I. R. et al., J. Clin. Invest. (2003) 111:1771-1782), therefore suggesting that the ubiquitin-proteasome machineryregulates osteoblast differentiation and bone formation. Therefore, thedisclosed compounds may be useful in the treatment and/or prevention ofdiseases associated with bone loss, such as osteoporosis.

Bone tissue is an excellent source for factors which have the capacityfor stimulating bone cells. Thus, extracts of bovine bone tissue containnot only structural proteins which are responsible for maintaining thestructural integrity of bone, but also biologically active bone growthfactors which can stimulate bone cells to proliferate. Among theselatter factors are a recently described family of proteins called bonemorphogenetic proteins (BMPs). All of these growth factors have effectson other types of cells, as well as on bone cells, including Hardy, M.H., et al., Trans Genet (1992) 8:55-61 describes evidence that bonemorphogenetic proteins (BMPs), are differentially expressed in hairfollicles during development. Harris, S. E., et al., J Bone Miner Res(1994) 9:855-863 describes the effects of TGF-β on expression of BMP-2and other substances in bone cells. BMP-2 expression in mature folliclesalso occurs during maturation and after the period of cell proliferation(Hardy, et al. (1992, supra). Thus, compounds of the invention may alsobe useful for hair follicle growth stimulation.

Finally, the disclosed compounds are also useful as diagnostic agents(e.g., in diagnostic kits or for use in clinical laboratories) forscreening for proteins (e.g., enzymes, transcription factors) processedby Ntn hydrolases, including the proteasome. The disclosed compounds arealso useful as research reagents for specifically binding the X/MB1subunit or α-chain and inhibiting the proteolytic activities associatedwith it. For example, the activity of (and specific inhibitors of) othersubunits of the proteasome can be determined.

Most cellular proteins are subject to proteolytic processing duringmaturation or activation. Enzyme inhibitors disclosed herein can be usedto determine whether a cellular, developmental, or physiological processor output is regulated by the proteolytic activity of a particular Ntnhydrolase. One such method includes obtaining an organism, an intactcell preparation, or a cell extract; exposing the organism, cellpreparation, or cell extract to a compound disclosed herein; exposingthe compound-exposed organism, cell preparation, or cell extract to asignal, and monitoring the process or output. The high selectivity ofthe compounds disclosed herein permits rapid and accurate elimination orimplication of the Ntn (for example, the 20S proteasome) in a givencellular, developmental, or physiological process.

Administration

Compounds prepared as described herein can be administered in variousforms, depending on the disorder to be treated and the age, condition,and body weight of the patient, as is well known in the art. Forexample, where the compounds are to be administered orally, they may beformulated as tablets, capsules, granules, powders, or syrups; or forparenteral administration, they may be formulated as injections(intravenous, intramuscular, or subcutaneous), drop infusionpreparations, or suppositories. For application by the ophthalmic mucousmembrane route, they may be formulated as eye drops or eye ointments.These formulations can be prepared by conventional means, and ifdesired, the active ingredient may be mixed with any conventionaladditive or excipient, such as a binder, a disintegrating agent, alubricant, a corrigent, a solubilizing agent, a suspension aid, anemulsifying agent, a coating agent, a cyclodextrin, and/or a buffer.Although the dosage will vary depending on the symptoms, age and bodyweight of the patient, the nature and severity of the disorder to betreated or prevented, the route of administration and the form of thedrug, in general, a daily dosage of from 0.01 to 2000 mg of the compoundis recommended for an adult human patient, and this may be administeredin a single dose or in divided doses. The amount of active ingredientwhich can be combined with a carrier material to produce a single dosageform will generally be that amount of the compound which produces atherapeutic effect.

The precise time of administration and/or amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given patient will depend upon the activity, pharmacokinetics, andbioavailability of a particular compound, physiological condition of thepatient (including age, sex, disease type and stage, general physicalcondition, responsiveness to a given dosage, and type of medication),route of administration, etc. However, the above guidelines can be usedas the basis for fine-tuning the treatment, e.g., determining theoptimum time and/or amount of administration, which will require no morethan routine experimentation consisting of monitoring the subject andadjusting the dosage and/or timing.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose ligands, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose, and sucrose; (2) starches, such as corn starch, potatostarch, and substituted or unsubstituted β-cyclodextrin; (3) cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6)gelatin; (7) talc; (8) excipients, such as cocoa butter and suppositorywaxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil,sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such aspropylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol,and polyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations. In certainembodiments, pharmaceutical compositions of the present invention arenon-pyrogenic, i.e., do not induce significant temperature elevationswhen administered to a patient.

The term “pharmaceutically acceptable salt” refers to the relativelynon-toxic, inorganic and organic acid addition salts of theinhibitor(s). These salts can be prepared in situ during the finalisolation and purification of the inhibitor(s), or by separatelyreacting a purified inhibitor(s) in its free base form with a suitableorganic or inorganic acid, and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate,stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, naphthylate, mesylate,glucoheptonate, lactobionate, laurylsulphonate salts, and amino acidsalts, and the like. (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66: 1-19.)

In other cases, the inhibitors useful in the methods of the presentinvention may contain one or more acidic functional groups and, thus,are capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable bases. The term “pharmaceutically acceptablesalts” in these instances refers to the relatively non-toxic inorganicand organic base addition salts of an inhibitor(s). These salts canlikewise be prepared in situ during the final isolation and purificationof the inhibitor(s), or by separately reacting the purified inhibitor(s)in its free acid form with a suitable base, such as the hydroxide,carbonate, or bicarbonate of a pharmaceutically acceptable metal cation,with ammonia, or with a pharmaceutically acceptable organic primary,secondary, or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts, and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like(see, for example, Berge et al., supra).

Wetting agents, emulsifiers, and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring, and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like;(2) oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert matrix, such as gelatin and glycerin, orsucrose and acacia) and/or as mouthwashes, and the like, each containinga predetermined amount of an inhibitor(s) as an active ingredient. Acomposition may also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), the active ingredient ismixed with one or more pharmaceutically acceptable carriers, such assodium citrate or dicalcium phosphate, and/or any of the following: (1)fillers or extenders, such as starches, cyclodextrins, lactose, sucrose,glucose, mannitol, and/or silicic acid; (2) binders, such as, forexample, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, acetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets, and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols, andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered inhibitor(s)moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pills,and granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes, and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents, and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor, and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols, and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active inhibitor(s) may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more inhibitor(s)with one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, which is solid at room temperature, butliquid at body temperature and, therefore, will melt in the rectum orvaginal cavity and release the active agent.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams, or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of aninhibitor(s) include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches, and inhalants. The active componentmay be mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams, and gels may contain, in addition toinhibitor(s), excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, andzinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to an inhibitor(s),excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

The inhibitor(s) can be alternatively administered by aerosol. This isaccomplished by preparing an aqueous aerosol, liposomal preparation, orsolid particles containing the composition. A nonaqueous (e.g.,fluorocarbon propellant) suspension could be used. Sonic nebulizers arepreferred because they minimize exposing the agent to shear, which canresult in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular composition,but typically include nonionic surfactants (Tweens, Pluronics, sorbitanesters, lecithin, Cremophors), pharmaceutically acceptable co-solventssuch as polyethylene glycol, innocuous proteins like serum albumin,oleic acid, amino acids such as glycine, buffers, salts, sugars, orsugar alcohols. Aerosols generally are prepared from isotonic solutions.

Transdermal patches have the added advantage of providing controlleddelivery of an inhibitor(s) to the body. Such dosage forms can be madeby dissolving or dispersing the agent in the proper medium. Absorptionenhancers can also be used to increase the flux of the inhibitor(s)across the skin. The rate of such flux can be controlled by eitherproviding a rate controlling membrane or dispersing the inhibitor(s) ina polymer matrix or gel.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more inhibitors(s) in combination withone or more pharmaceutically acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents, and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include tonicity-adjusting agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. For example, delayed absorption of a parenterallyadministered drug form is accomplished by dissolving or suspending thedrug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofinhibitor(s) in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

The preparations of agents may be given orally, parenterally, topically,or rectally. They are, of course, given by forms suitable for eachadministration route. For example, they are administered in tablets orcapsule form, by injection, inhalation, eye lotion, ointment,suppository, infusion; topically by lotion or ointment; and rectally bysuppositories. Oral administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection, and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a ligand, drug, or other materialother than directly into the central nervous system, such that it entersthe patient's system and thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These inhibitors(s) may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally, and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the inhibitor(s),which may be used in a suitable hydrated form, and/or the pharmaceuticalcompositions of the present invention, are formulated intopharmaceutically acceptable dosage forms by conventional methods knownto those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The concentration of a disclosed compound in a pharmaceuticallyacceptable mixture will vary depending on several factors, including thedosage of the compound to be administered, the pharmacokineticcharacteristics of the compound(s) employed, and the route ofadministration. In general, the compositions of this invention may beprovided in an aqueous solution containing about 0.1-10% w/v of acompound disclosed herein, among other substances, for parenteraladministration. Typical dose ranges are from about 0.01 to about 50mg/kg of body weight per day, given in 1-4 divided doses. Each divideddose may contain the same or different compounds of the invention. Thedosage will be an effective amount depending on several factorsincluding the overall health of a patient, and the formulation and routeof administration of the selected compound(s).

Another aspect of the invention provides a conjoint therapy wherein oneor more other therapeutic agents are administered with the proteasomeinhibitor. Such conjoint treatment may be achieved by way of thesimultaneous, sequential, or separate dosing of the individualcomponents of the treatment.

In certain embodiments, a compound of the invention is conjointlyadministered with one or more other proteasome inhibitor(s).

In certain embodiments, a compound of the invention is conjointlyadministered with a chemotherapeutic. Suitable chemotherapeutics mayinclude, natural products such as vinca alkaloids (i.e. vinblastine,vincristine, and vinorelbine), paclitaxel, epidipodophyllotoxins (i.e.etoposide, teniposide), antibiotics (dactinomycin (actinomycin D)daunorubicin, doxorubicin and idarubicin), anthracyclines, mitoxantrone,bleomycins, plicamycin (mithramycin) and mitomycin, enzymes(L-asparaginase which systemically metabolizes L-asparagine and deprivescells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates(busulfan), nitrosoureas (carmustine (BCNU) and analogs, streptozocin),trazenes—dacarbazinine (DTIC); antiproliferative/antimitoticantimetabolites such as folic acid analogs (methotrexate), pyrimidineanalogs (fluorouracil, floxuridine, and cytarabine), purine analogs andrelated inhibitors (mercaptopurine, thioguanine, pentostatin and2-chlorodeoxyadenosine); aromatase inhibitors (anastrozole, exemestane,and letrozole); and platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;histone deacetylase (HDAC) inhibitors (trichostatin, sodium butyrate,apicidan, suberoyl anilide hydroamic acid); hormones (i.e. estrogen) andhormone agonists such as leutinizing hormone releasing hormone (LHRH)agonists (goserelin, leuprolide and triptorelin). Other chemotherapeuticagents may include mechlorethamine, camptothecin, ifosfamide, tamoxifen,raloxifene, gemcitabine, navelbine, or any analog or derivative variantof the foregoing.

In certain embodiments, a compound of the invention is conjointlyadministered with a cytokine. Cytokines include, but are not limited to,Interferon-γ, -α, and -β, Interleukins 1-8, 10 and 12, GranulocyteMonocyte Colony-Stimulating factor (GM-CSF), TNF-α and -β, and TGF-β.

In certain embodiments, a compound of the invention is conjointlyadministered with a steroid. Suitable steroids may include, but are notlimited to, 21-acetoxypregnenolone, alclometasone, algestone,amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone,clobetasol, clocortolone, cloprednol, corticosterone, cortisone,cortivazol, deflazacort, desonide, desoximetasone, dexamethasone,diflorasone, diflucortolone, difuprednate, enoxolone, fluazacort,flucloronide, flumethasone, flunisolide, fluocinolone acetonide,fluocinonide, fluocortin butyl, fluocortolone, fluorometholone,fluperolone acetate, fluprednidene acetate, fluprednisolone,flurandrenolide, fluticasone propionate, formocortal, halcinonide,halobetasol propionate, halometasone, hydrocortisone, loteprednoletabonate, mazipredone, medrysone, meprednisone, methylprednisolone,mometasone furoate, paramethasone, prednicarbate, prednisolone,prednisolone 25-diethylaminoacetate, prednisolone sodium phosphate,prednisone, prednival, prednylidene, rimexolone, tixocortol,triamcinolone, triamcinolone acetonide, triamcinolone benetonide,triamcinolone hexacetonide, and salts and/or derivatives thereof.

In certain embodiments, a compound of the invention is conjointlyadministered with an immunotherapeutic agent. Suitable immunotherapeuticagents may include, but are not limited to, MDR modulators (verapamil,valspordar, biricodar, tariquidar, laniquidar), cyclosporine,thalidomide, and monoclonal antibodies. The monoclonal antibodies can beeither naked or conjugated such as rituximab, tositumomab, alemtuzumab,epratuzumab, ibritumomab tiuxetan, gemtuzumab ozogamicin, bevacizumab,cetuximab, erlotinib and trastuzumab.

Exemplification

Synthesis of (A)

To a solution of NBoc leucine (19.81 g, 85.67 mmol, 1.0 eq.) andphenylalanine benzyl ester (25.0 g, 85.67 mmol, 1.0 eq.) in 900 mL ofMeCN was added DIEA (44.29 g, 60 mL, 342.68 mmol, 4.0 eq.) and themixture was cooled to 0° C. in an ice bath. To this mixture was addedHOBT (18.52 g, 137.08 mmol, 1.6 eq) followed by PyBOP (71.33 g, 137.08mmol, 1.6 eq) which was added in several portions over five minutes. Thereaction was placed under an atmosphere of argon and stirred overnight.The volatiles were removed under reduced pressure and the remainingmaterial was taken up in 500 mL of EtOAc and washed with sat. NaHCO₃,H₂O, and brine and dried over MgSO₄. The MgSO₄ was removed by filtrationand the volatiles removed under reduced pressure. To a 0° C. cooledsolution of 70% TFA/DCM (150 mL) was added BocNHLeuPheOBz (25.0 g, 53.35mmol, 1.0 eq.). The solution was stirred and allowed to warm to roomtemperature over 2 hr at which time the mixture was concentrated andplaced under high vacuum for 2 hours giving the TFA salt of thedi-peptide amine. To the resulting oil was added BocNHhPheCO₂H (14.68 g,53.35 mmol, 1.0 eq.), 550 mL of MeCN, and DIEA (27.58 g, 37.2 mL, 213.4mmol, 4.0 eq.) and the mixture was cooled to 0° C. in an ice bath. Tothe cooled mixture was added HOBT (11.53 g, 85.36 mmol, 1.6 eq.)followed by PyBOP (44.42 g, 85.36 mmol, 1.6 eq.) which was added inseveral portions over five minutes. The reaction was placed under argonand allowed to warm to room temperature overnight at which time a whiteprecipitate had formed. The reaction mixture was cooled and the solidswere collected by filtration and then washed with cold MeCN to give (A)(24.86 g).

Synthesis of (B)

Intermediate (A) (23.0 mmol, 14.5 g) was mixed with TFA/DCM (80%) andstirred at room temperature for one hour at which time the mixture wasconcentrated and placed under high vacuum for 2 hours giving (B).

Synthesis of (C)

To a solution of (B) (1.6 mmol, 1 eq.) in MeCN (100 mL) was added5-chlorovaleryl chloride (1.9 mmol, 0.24 mL, 1.2 eq.) and DIEA (6.4mmol, 1.2 mL, 4 eq.). The mixture was stirred at room temperatureovernight and then concentrated to give a solid. The solid was collectedand washed with ether to give the alkyl chloride. To a solution of thealkyl chloride (0.21 mmol, 0.134 g) in dry acetone (100 mL) was addedNaI (2.5 mmol, 0.387 g) and the reaction was refluxed overnight. Thereaction mixture was then concentrated under vacuum and the residuedissolved in EtOAc, washed with water and brine, and dried over MgSO₄.The MgSO₄ was removed by filtration and the volatiles removed underreduced pressure giving (C).

Synthesis of (D)

To a solution of (C) (0.040 mmol, 30.0 mg) in THF (2 mL) was addedpiperidine (0.048 mmol, 5.0 mg) and DIEA (0.040 mmol, 0.5 mg). Afterstirring for 2 hours at room temperature, the contents were concentratedand dissolved in EtOAc, washed with water, brine, and dried over MgSO₄.The MgSO₄ was removed by filtration and the volatiles removed underreduced pressure. The crude ester was dissolved in 1:1 EtOAc/MeOH (10mL), 5% Pd/C (30.0 mg) was added, and the mixture placed under 1atmosphere of hydrogen for 2 hours. The reaction was filtered throughCelite and the volatiles removed under reduced pressure affording (D)(11.0 mg).

Synthesis of Compound 1

To a stirred solution of (E) [see: Bioorg. Med. Chem. Lett., 1999, 9,2283-2288] (0.098 mmol, 5.2 eq.) in DMF (3 mL) was added (D) (0.019mmol, 0.014 g, 1 eq.), DIEA (0.50 mmol, 88 μL, 20 eq.), and HOBT (0.20mmol, 0.0272 g, 10.5 eq.). The mixture was cooled to 0° C. in an icebath and PyBOP (0.20 mmol, 0.105 g, 10.5 eq.) was added in severalportions. The mixture was then stirred at 5° C. under an atmosphere ofnitrogen overnight. The reaction was then diluted with sat. NaCl andextracted with EtOAc. The organic layer was washed with water and brine,dried over anhydrous MgSO₄, and concentrated to an oil that was purifiedby flash chromatography to afford compound 1 (5.1 mg). IC₅₀ 20 S CT-L<50nM, IC₅₀ Cell-based CT-L<50 nM.

Synthesis of (F)

To a solution of (C) (0.040 mmol, 0.030 g) in THF (2 mL) was addedmorpholine (0.050 mmol, 5.0 mg) and DIEA (0.040 mmol, 0.5 mg). Afterstirring 2 hours at room temperature the contents were concentrated anddissolved in EtOAc, washed with water and brine, and dried over MgSO₄.The MgSO₄ was removed by filtration and the volatiles removed underreduced pressure. The crude ester was dissolved in 1:1 EtOAc/MeOH (10mL), 5% Pd/C (30.0 mg) added, and the mixture placed under 1 atmosphereof hydrogen for 2 hours. The reaction was filtered through Celite andthe volatiles removed under reduced pressure affording (F) (19.0 mg).

Synthesis of Compound 2

To a stirred solution of (E) [see: Bioorg. Med. Chem. Lett., 1999, 9,2283-2288] (0.098 mmol, 3.2 eq.) in DMF (3 mL) was added (F) (0.030mmol, 0.018 g, 1 eq.), DIEA (0.50 mmol, 88 μL, 17 eq.), and HOBT (0.20mmol, 27.2 mg, 6.7 eq.). The mixture was cooled to 0° C. in an ice bathand PyBOP (0.20 mmol, 0.105 g, 6.7 eq.) was added in several portions.The mixture was then stirred at 5° C. under an atmosphere of nitrogenovernight. The reaction was then diluted with sat. NaCl and extractedwith EtOAc. The organic layer was washed with water and brine, driedover anhydrous MgSO₄, and concentrated to an oil that was purified byflash chromatography to afford compound 2 (6.0 mg). IC₅₀ 20 S CT-L<50nM, IC₅₀ Cell-based CT-L<50 nM.

Synthesis of (G)

To a solution of (C) (0.040 mmol, 30.0 mg) in THF (2 mL) was addedN-methylpiperazine (0.050 mmol, 5.0 mg) and DIEA (0.040 mmol, 0.5 mg).After stirring for 2 hours at room temperature, the contents wereconcentrated and dissolved in EtOAc, washed with water and brine, anddried over MgSO₄. The MgSO₄ was removed by filtration and the volatilesremoved under reduced pressure. The crude ester was dissolved in 1:1EtOAc/MeOH (10 mL), 5% Pd/C (30.0 mg) added and the mixture was placedunder 1 atmosphere of hydrogen for 2 hours. The reaction was filteredthrough Celite and the volatiles removed under reduced pressureaffording (G) (31.0 mg).

Synthesis of Compound 3

To a stirred solution of (E) [see: Bioorg. Med. Chem. Lett, 1999, 9,2283-2288] (0.098 mmol, 3.2 eq.) in DMF (3 mL) was added (G) (0.030mmol, 18.0 mg, 1 eq.), DIEA (0.50 mmol, 88 μL, 17 eq.), and HOBT (0.20mmol, 27.2 mg, 6.7 eq.). The mixture was cooled to 0° C. in an ice bathand PyBOP (0.20 mmol, 0.105 g, 6.7 eq.) was added in several portions.The mixture was then stirred at 5° C. under an atmosphere of nitrogenovernight. The reaction was then diluted with sat. NaCl and extractedwith EtOAc. The organic layer was washed with water and brine, driedover anhydrous MgSO₄, and concentrated to an oil that was purified byflash chromatography to afford compound 3 (3.9 mg). IC₅₀ 20 S CT-L<50nM, IC₅₀ Cell-based CT-L<50 nM.

Synthesis of (I)

To a solution of (B) (2.0 mmol, 1 eq.) in MeCN (120 mL) was added4-chlorobutryl chloride (2.8 mmol, 0.32 mL, 1.2 eq.) and DIEA (8 mmol,1.4 mL, 4 eq.). The mixture was stirred at room temperature overnightand then concentrated to give a solid. The solid was collected andwashed with ether to give the alkyl chloride (0.808 g). To a solution ofthe alkyl chloride (0.09 mmol, 0.060 g) in dry acetone (10 mL) was addedNaI (0.86 mmol, 0.130 g) and the reaction was refluxed overnight. Thecontents were concentrated under vacuum and the residue dissolved inDCM, washed with water and brine, and dried over MgSO₄. The MgSO₄ wasremoved by filtration and the volatiles removed under reduced pressure.Purification by flash chromatography afforded (I) (0.050 g).

Synthesis of (J)

To a solution of (I) (0.040 mmol, 30.0 mg) in THF (2 mL) was addedpiperidine (0.050 mmol, 4.0 mg) and DIEA (0.040 mmol, 0.5 mg). Afterstirring overnight at room temperature, the contents were concentratedand dissolved in EtOAc, washed with water and brine, and dried overMgSO₄. The MgSO₄ was removed by filtration and the volatiles removedunder reduced pressure. The crude ester was dissolved in 1:1 EtOAc/MeOH(10 mL), 5% Pd/C (0.020 g) added, and the mixture placed under 1atmosphere of hydrogen for 2 hours. The reaction was filtered throughCelite and the volatiles removed under reduced pressure affording (J).

Synthesis of Compound 5

To a stirred solution of (E) [see: Bioorg. Med. Chem. Lett., 1999, 9,2283-2288] (0.098 mmol, 4.9 eq.) in DMF (3 mL) was added (J) (0.020mmol, 1 eq.), DIEA (0.18 mmol, 31 μL, 9 eq.), and HOBT (0.074 mmol, 10.0mg, 3.7 eq.). The mixture was cooled to 0° C. in an ice bath and PyBOP(0.07 mmol, 36.0 mg, 3.7 eq.) was added in several portions. The mixturewas stirred at 5° C. under an atmosphere of nitrogen overnight. Thereaction was then diluted with sat. NaCl and extracted with EtOAc. Theorganic layer was washed with water and brine, dried over anhydrousMgSO₄, and concentrated to an oil that was purified by flashchromatography to afford compound 5 (18.2 mg). IC₅₀ 20 S CT-L<50 nM,IC₅₀ Cell-based CT-L<50 nM.

Synthesis of (K)

To a solution of (I) (0.040 mmol, 30.0 mg) in THF (2 mL) was addedmorpholine (0.050 mmol, 5.0 mg) and DIEA (0.040 mmol, 0.5 mg). Afterstirring overnight at room temperature, the contents were concentrated,dissolved in EtOAc, washed with water and brine, and dried over MgSO₄.The MgSO₄ was removed by filtration and the volatiles were removed underreduced pressure. The crude ester was dissolved in 1:1 EtOAc/MeOH (10mL), 5% Pd/C (20.0 mg) added, and the mixture placed under 1 atmosphereof hydrogen for 2 hours. The reaction was filtered through Celite andthe volatiles removed under reduced pressure affording (K).

Synthesis of Compound 6

To a stirred solution of (E) [see: Bioorg. Med. Chem. Lett., 1999, 9,2283-2288] (0.151 mmol, 1.2 eq.) in DMF (3 mL) was added (K) (0.126mmol, 0.075 g, 1 eq.), DIEA (0.50 mmol, 88 μL, 4 eq.), and HOBT (0.20mmol, 27.0 mg, 1.6 eq.). The mixture was cooled to 0° C. in an ice bathand PyBOP (0.202 mmol, 0.105 g, 1.6 eq.) was added in several portions.The mixture was stirred at 5° C. under an atmosphere of nitrogenovernight. The reaction was then diluted with sat. NaCl and extractedwith EtOAc. The organic layer was washed with water and brine, driedover anhydrous MgSO₄, and concentrated to an oil that was purified byflash chromatography to afford compound 6 (46.6 mg). IC₅₀ 20 S CT-L<50nM, IC₅₀ Cell-based CT-L<50 nM.

Synthesis of (L)

To a solution of (1) (0.040 mmol, 30.0 mg) in THF (2 mL) was addedN-methylpiperazine (0.050 mmol, 5.0 mg) and DIEA (0.040 mmol, 0.5 mg).After stirring overnight at room temperature the contents wereconcentrated and dissolved in EtOAc, washed with water and brine, anddried over MgSO₄. The MgSO₄ was removed by filtration and the volatilesremoved under reduced pressure. The crude ester was dissolved in 1:1EtOAc/MeOH (10 mL), 5% Pd/C (20.0 mg) added, and the mixture placedunder 1 atmosphere of hydrogen for 2 hours. The reaction was filteredthrough Celite and the volatiles removed under reduced pressureaffording (L).

Synthesis of Compound 7

To a stirred solution of (E) [see: Bioorg. Med. Chem. Lett., 1999, 9,2283-2288] (0.098 mmol, 1.5 eq.) in DMF (3 mL) was added (L) (0.065mmol, 0.075 g, 1 eq.), DIEA (0.50 mmol, 88 μL, 8 eq.), and HOBT (0.20mmol, 27.0 mg, 3.1 eq.). The mixture was cooled to 0° C. in an ice bathand PyBOP (0.20 mmol, 0.105 g, 3.1 eq.) was added in several portions.The mixture was then stirred at 5° C. under an atmosphere of nitrogenovernight. The reaction was then diluted with sat. NaCl and extractedwith EtOAc. The organic layer was washed with water and brine, driedover anhydrous MgSO₄, and concentrated to an oil that was purified byflash chromatography to afford compound 7 (4.8 mg). IC₅₀ 20 S CT-L<50nM, IC₅₀ Cell-based CT-L<50 nM.

Synthesis of (N)

Compound (B) (0.39 mmol) was dissolved in DMF (6 mL) and4-morpholinoacetic acid (0.507 mmol, 0.074 g) was added followed by DIEA(3.90 mmol, 0.504 g, 0.68 mL). The mixture was cooled to 0° C. in an icebath and PyBOP (0.62 mmol, 0.32 g) was added and stirred under anatmosphere of argon while warming to room temperature overnight. Themixture was diluted with brine (50 mL) and extracted with EtOAc (5×20mL). The organic layers were combined, washed with sat. NaHCO₃ (5×15 mL)and brine (1×25 mL), and dried over MgSO₄. The MgSO₄ was removed byfiltration and the volatiles removed under reduced pressure to give theintermediate ester (M) (0.195 g). To (M) (0.150 g, 0.23 mmol) was added10% Pd/C (0.05 g) followed by 5 mL of 1:1 mixture of MeOH and EtOAc andthe mixture was placed under an atmosphere of hydrogen. After 2 hr, thecontents were filtered through a plug of Celite and concentrated undervacuum to give (N) (0.12 g).

Synthesis of Compound 8

To a stirred solution of (E) [see: Bioorg. Med. Chem. Lett., 1999, 9,2283-2288] (0.27 mmol, 0.083 mg, 1.3 eq.) in MeCN (5 mL) was added (N)(0.17 mmol, 0.10 g, 1 eq.), DIEA (1.73 mmol, 0.30 mL, 10 eq.) and HOBT(0.27 mmol, 0.037 mg, 1.6 eq.). The mixture was cooled to 0° C. in anice bath and PyBOP (0.27 mmol, 0.14 g, 1.6 eq.) was added in severalportions. The mixture was stirred at 5° C. under an atmosphere of argonovernight after which, the reaction was diluted with sat. NaCl andextracted with EtOAc. The organic layer was washed with water and brine,dried over anhydrous MgSO₄, and concentrated to a paste. The crudematerial was dissolved in a minimum amount of MeOH and slowly added intorapidly stirred, 0° C. chilled water (100 mL). Compound 8 was thenisolated by filtration (0.080 g). IC₅₀ 20 S CT-L<50 nM, IC₅₀ Cell-basedCT-L<50 nM.

Synthesis of (P)

To a 0° C. solution of (0) [prepared by following the same procedure forthe synthesis of (B) except substituting phenylalanine methyl ester forphenylalanine benzyl ester] (1.8 mmol, 1 eq.) in DMF (10 mL) was addedchloroacetyl chloride (2.7 mmol, 0.22 mL, 1.5 eq.) and DIEA (3.5 mmol,1.4 mL, 3 eq.). The mixture was allowed to warm and stirred at roomtemperature overnight. The reaction was concentrated under vacuum anddissolved in EtOAc, washed with water and brine, and dried over Na₂SO₄.The Na₂SO₄ was removed by filtration and the volatiles removed underreduced pressure to afford (P) (0.64 g).

Synthesis of (Q)

To a solution of (P) (0.188 mmol, 0.10 g) in THF (20 mL) was addedN-methylpiperazine (0.226 mmol, 22.0 mg) and KI (0.04 mmol, 6.4 mg).After stirring overnight at room temperature the contents wereconcentrated and dissolved in EtOAc, washed with water and brine, anddried over MgSO₄. The MgSO₄ was removed by filtration and the volatilesremoved under reduced pressure giving the crude ester (0.095 g). Thecrude ester (0.095 g) was dissolved in 3:1 MeOH/H₂O (8 mL), cooled to 0°C., and LiOH (1.6 mmol, 39.0 mg) was added. The mixture was stirred at5° C. overnight, quenched with sat. NH₄Cl, diluted with water (20 mL),and the pH adjusted to 3 with 1N HCl. The mixture was extracted withchloroform and the organic layers combined and dried over Na₂SO₄. TheNa₂SO₄ was removed by filtration and the volatiles removed under reducedpressure to afford (Q) (20.0 mg).

Synthesis of Compound 9

To a stirred solution of (E) [see: Bioorg. Med. Chem. Lett., 1999, 9,2283-2288] (0.082 mmol, 2.4 eq.) in DMF (1 mL) was added (Q) (0.034mmol, 0.075 g, 1 eq.), DIEA (0.29 mmol, 50 μL, 8.5 eq.), and HOBT (0.13mmol, 18.0 mg, 3.8 eq.). The mixture was cooled to 0° C. in an ice bathand BOP (0.13 mmol, 0.058 g, 3.8 eq.) was added in several portions. Themixture was then stirred at 5° C. under an atmosphere of nitrogenovernight. The reaction was then diluted with sat. NaCl and extractedwith EtOAc. The organic layer was washed with water and brine, driedover anhydrous MgSO₄, filtered, and concentrated to an oil that waspurified by flash chromatography to afford compound 9. IC₅₀ 20 S CT-L<50nM, IC₅₀ Cell-based CT-L<50 nM.

Synthesis of (R)

To a solution of benzyl 2-bromoacetate (4.56 mmol, 0.715 mL) and4-(2-hydroxyethyl)morpholine (3.8 mmol, 0.466 mL) in DMF (4 mL) wasadded NaH (5.7 mmol, 0.136 g) and the mixture stirred overnight under anatmosphere of nitrogen. The reaction was diluted with brine andextracted with EtOAc. The organic layers were combined, washed withwater and brine, and dried over MgSO₄. The MgSO₄ was removed byfiltration and the volatiles removed under reduced pressure. The crudeester was purified by flash chromatography. The purified ester wasdissolved in 1:1 MeOH/EtOAc (10 mL), 5% Pd/C (0.100 g) was added, andthe mixture placed under an atmosphere of hydrogen overnight. Thereaction was purged, filtered through Celite and concentrated undervacuum affording (R) (0.107 g).

Synthesis of (S)

To a solution of (B) (0.56 mmol) in DMF (15 mL), compound (R) (0.56mmol, 0.107 g) was added followed by DIEA (2.24 mmol, 0.391 mL). Themixture was cooled to 0° C. in an ice bath and HOBT (0.90 mmol, 0.121 g)and PyBOP (0.90 mmol, 0.466 g) were added and the reaction was stirredunder an atmosphere of argon while warming to room temperatureovernight. The mixture was diluted with brine (50 mL) and extracted withEtOAc (5×20 mL). The organic layers were combined, washed with sat.NaHCO₃ (5×15 mL) and brine (1×25 mL), and dried over MgSO₄. The MgSO₄was removed by filtration and the volatiles removed under reducedpressure to give (S).

Synthesis of (T)

To a solution of (S) (0.56 mmol) in 1:1 MeOH/EtOAc (10 mL) was added 5%Pd/C (0.1 g) and the mixture placed under an atmosphere of hydrogenovernight. The reaction was purged, filtered through Celite andconcentrated under vacuum to give (T).

Synthesis of Compound 10

To a stirred solution of (E) [see: Bioorg. Med. Chem. Lett., 1999, 9,2283-2288] (0.164 mmol, 1.0 eq.) in DMF (10 mL) was added (T) (0.16mmol, 0.100 g, 1 eq.), DIEA (0.64 mmol, 112 μL, 4.0 eq.), and HOBT (0.25mmol, 35.0 mg, 1.6 eq.). The mixture was cooled to 0° C. in an ice bathand PyBOP (0.25 mmol, 0.133 g, 1.6 eq.) was added in several portions.The mixture was stirred at 5° C. under an atmosphere of nitrogenovernight. The reaction was then diluted with sat. NaCl and extractedwith EtOAc. The organic layer was washed with water and brine, driedover anhydrous MgSO₄, and concentrated to an oil that was purified byflash chromatography to afford compound 10 (19.0 mg). IC₅₀ 20 S CT-L<50nM, IC₅₀ Cell-based CT-L<50 nM.

Synthesis of (W)

To a solution of Fmoc-Phe (4-CF₃)—OH (2.2 mmol, 1.0 g) in DCM (20 mL)was added 1-methylimidizole (6.7 mmol, 0.370 mL). When the solution washomogeneous, 1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT)(2.9 mmol, 0.870 g) was added. Once the MSNT dissolved, the reactionmixture was added to Wang resin (0.8 mmol, 1.0 g) and the resultingsolution was allowed to shake for 45 minutes. The resin was filtered andwashed with DMF (50 mL), MeOH (50 mL), and DCM (50 mL). The resultingresin was allowed to air dry, to yield (W).

Synthesis of (X)

To (W) (0.40 mmol, 0.5 g) was added 20% piperidine/DMF (10 mL) and theresulting heterogeneous solution was allowed to shake for 20 minutes.The mixture was filtered and the resin washed with DMF (20 mL), MeOH (20mL), and DCM (20 mL) and allowed to air dry. The resin was subjected tothe above reaction condition a second time to yield (X).

Synthesis of (Y)

To (X) (0.40 mmol) was added DMF (20 mL), Fmoc-Leu-OH (0.40 mmol, 0.143g), DIEA (1.6 mmol, 0.12 mL), HOBT (0.64 mmol, 0.086 g), and BOP (0.64mmol, 0.178 g) and the reaction mixture was allowed to shake overnight.The reaction mixture was filtered and the resin washed with DMF (40 mL),MeOH (40 mL), and DCM (40 mL), and allowed to air dry, to yield (Y).

Synthesis of (Z)

To (Y) (0.08 mmol, 0.10 g) was added 20% piperidine/DMF (2 mL) and theresulting heterogeneous solution was allowed to shake for 20 minutes.The solution was filtered and the resin washed with DMF (10 mL), MeOH(10 mL), and DCM (10 mL) and allowed to air dry. The resin was subjectedto the above reaction condition a second time to yield (Z).

Synthesis of (AA)

To (Z) (0.08 mmol, 0.10 g) was added DMF (20 mL), Fmoc-hPhe-OH (0.40mmol, 0.143 g), DIEA (1.6 mmol, 0.12 mL), HOBT (0.64 mmol, 0.062 mg),and BOP (0.64 mmol, 0.178 g) and the reaction mixture was allowed toshake overnight. The reaction mixture was filtered and the resin washedwith DMF (40 mL), MeOH (40 mL), and DCM (40 mL), and allowed to air dry,to yield (AA).

Synthesis of (BB)

To (AA) (0.08 mmol, 0.10 g) was added 20% piperidine/DMF (2 mL) and theresulting heterogeneous solution was allowed to shake for 20 minutes.The solution was filtered and the resin washed with DMF (10 mL), MeOH(10 mL), and DCM (10 mL) and allowed to air dry. The resin was subjectedto the above reaction condition a second time, to yield (BB).

Synthesis of (CC)

To (BB) (0.08 mmol, 0.10 g) was added DMF (2 mL), 4-morpholinoaceticacid (0.10 mmol, 0.015 g), DIEA (0.17 mmol, 0.029 mL), HOBT (0.11 mmol,0.016 g), and BOP (0.11 mmol, 0.051 g) and the reaction mixture wasallowed to shake overnight. The reaction mixture was filtered and theresin washed with DMF (15 mL), MeOH (15 mL), and DCM (15 mL), andallowed to air dry, to yield (CC).

Synthesis of (DD)

To (CC) (0.08 mmol, 0.10 g) was added 50% TFA/DCM (2 mL) and the mixturewas allowed to shake for 20 minutes (the resin turned purple). Thereaction was filtered and the resin washed with DCM (10 mL). Thevolatiles were removed under reduced pressure and the resulting oil wasdiluted with DCM (10 mL) and evaporated a total of three times to yield(DD).

Synthesis of Compound 13

To a stirred solution of (E) [see: Bioorg. Med. Chem. Lett., 1999, 9,2283-2288] (0.11 mmol, 0.019 g) in MeCN (2 mL) was added (DD) (0.1mmol), DIEA (2.9 mmol, 0.5 mL), HOBT (0.2 mmol, 0.032 g), and BOP (0.23mmol, 0.103 g) and the mixture was stirred at room temperatureovernight. The reaction was diluted with brine (15 mL) and extractedwith EtOAc. The organic layer was washed with water, sat. NaHCO₃, andbrine and dried over anhydrous MgSO₄. The MgSO₄ was removed byfiltration and the volatiles removed under reduced pressure. The crudematerial was purified by flash chromatography to afford 13 (12.6 mg).IC₅₀ 20 S CT-L<500 nM, IC₅₀ Cell-based CT-L<50 nM.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thecompounds and methods of use thereof described herein. Such equivalentsare considered to be within the scope of this invention and are coveredby the following claims.

All of the above-cited references and publications are herebyincorporated by reference.

We claim:
 1. A compound having a structure of formula I or apharmaceutically acceptable salt thereof,

wherein X is O, NH, or N-alkyl; Y is NH, N-alkyl, O, or C(R⁹)₂; Z is Oor C(R⁹)₂; R¹, R², R³, and R⁴ are all hydrogen; each R⁵, R⁶, R⁷, R⁸, andR⁹ is independently selected from hydrogen, C₁₋₆alkyl, C₁₋₆hydroxyalkyl,C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, each of which is optionallysubstituted with a group selected from alkyl, amide, amine, carboxylicacid or a pharmaceutically acceptable salt thereof, carboxyl ester,thiol, and thioether; R¹⁰ and R¹¹ are independently selected fromhydrogen and C₁₋₆alkyl, or R¹⁰ and R¹¹ together form a 3- to 6-memberedcarbocyclic or heterocyclic ring; R¹² and R¹³ are independently selectedfrom hydrogen, a metal cation, C₁₋₆alkyl, and C₁₋₆aralkyl, or R¹² andR¹³ together represent C₁₋₆alkyl, thereby forming a ring; m is aninteger from 0 to 2; and n is an integer from 0 to 2, preferably 0 or 1.2. A compound of claim 1, wherein X is O.
 3. A compound of claim 2,wherein R⁵, R⁶, R⁷, and R⁸ are independently selected from C₁₋₆alkyl,C₁₋₆hydroxyalkyl, and C₁₋₆aralkyl; and R⁹ is hydrogen.
 4. A compound ofclaim 3, wherein R⁵ and R⁷ are independently C₁₋₆aralkyl and R⁶ and R⁸are independently C₁₋₆alkyl.
 5. A compound of claim 4, wherein R¹, R²,R³, and R⁴ are all hydrogen.
 6. A compound of claim 5, wherein Y isselected from N-alkyl, O, and CH₂.
 7. A compound of claim 6, wherein Zis CH₂, and m and n are both
 0. 8. A compound of claim 6, wherein Z isCH₂, m is O, and n is 2 or
 3. 9. A compound of claim 6, wherein Z is O,m is 1, and n is
 2. 10. A compound of claim 1, having the followingstructure


11. A compound of claim 1, having the following structure


12. A compound of claim 1, having the structure


13. A compound of claim 1, having the structure


14. A compound of claim 1, having the structure


15. A compound of claim 1, having the structure


16. A compound having a structure of formula III or a pharmaceuticallyacceptable salt thereof,

wherein X is selected from O, NH, and N-alkyl; R¹, R², R³, and R⁴ areall hydrogen; R⁵, R⁶, R⁷, and R⁸ are independently selected fromhydrogen, C₁₋₆alkyl, C₁₋₆ hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, andC₁₋₆aralkyl, each of which is optionally substituted with a groupselected from amide, amine, carboxylic acid or a pharmaceuticallyacceptable salt thereof, carboxyl ester, thiol, and thioether.
 17. Acompound of claim 16, wherein X is O.
 18. A compound of claim 17,wherein R⁵, R⁶, R⁷, and R⁸ are independently selected from C₁₋₆alkyl,C₁₋₆ hydroxyalkyl, and C₁₋₆aralkyl.
 19. A compound of claim 18, whereinR⁵ and R⁷ are independently C₁₋₆aralkyl and R⁶ and R⁸ are independentlyC₁₋₆alkyl.
 20. A compound having a structure of formula IV or apharmaceutically acceptable salt thereof,

wherein X is O, NH, or N-alkyl; R¹, R², R³, and R⁴ are all hydrogen; R⁶and R⁸ are independently selected from hydrogen, C₁₋₆alkyl,C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, each of whichis optionally substituted with a group selected from amide, amine,carboxylic acid or a pharmaceutically acceptable salt thereof, carboxylester, thiol, and thioether.
 21. A compound of claim 20, wherein X is O.22. A compound of claim 21, wherein R⁶ and R⁸ are independently selectedfrom C₁₋₆alkyl, C₁₋₆hydroxyalkyl, and C₁₋₆aralkyl.
 23. A compound ofclaim 22, wherein R⁶ and R⁸ are independently C₁₋₆alkyl.
 24. A compoundof claim 23, wherein R⁶ and R⁸ are both isobutyl.
 25. A compound ofclaim 20, having the following structure


26. A compound having a structure of formula V or a pharmaceuticallyacceptable salt thereof,

where X is O, NH, or N-alkyl; R¹, R², R³, and R⁴ are all hydrogen and;R⁵, R⁶, R⁷, and R⁸ are independently selected from hydrogen, C₁₋₆alkyl,C₁₋₆hydroxyalkyl, C₁₋₆ alkoxyalkyl, aryl, and C₁₋₆aralkyl, each of whichis optionally substituted with a group selected from amide, amine,carboxylic acid or a pharmaceutically acceptable salt thereof, carboxylester, thiol, and thioether; and q is an integer from 0 to
 3. 27. Acompound of claim 26, wherein X is O.
 28. A compound of claim 27,wherein R⁵, R⁶, R⁷, and R⁸ are independently selected from C₁₋₆alkyl,C₁₋₆hydroxyalkyl, and C₁₋₆aralkyl.
 29. A compound of claim 28, whereinR⁵ and R⁷ are independently C₁₋₆aralkyl and R⁶ and R⁸ are independentlyC₁₋₆alkyl.
 30. A compound having a structure of formula VI or apharmaceutically acceptable salt thereof,

wherein X is O, NH, or N-alkyl; R¹, R², R³, and R⁴ are all hydrogen; R⁶and R⁸ are independently selected from hydrogen, C₁₋₆alkyl,C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, each of whichis optionally substituted with a group selected from amide, amine,carboxylic acid or a pharmaceutically acceptable salt thereof, carboxylester, thiol, and thioether; and q is an integer from 0 to
 3. 31. Acompound of claim 30, wherein X is O.
 32. A compound of claim 31,wherein R⁶ and R⁸ are independently selected from C₁₋₆alkyl,C₁₋₆hydroxyalkyl, and C₁₋₆aralkyl.
 33. A compound of claim 32, whereinR⁶ and R⁸ are independently C₁₋₆alkyl.
 34. A compound of claim 33,wherein R⁶ and R⁸ are both isobutyl.
 35. A compound of claim 30, havingthe structure


36. A compound of claim 30, having the structure


37. A compound of claim 30, having the following structure


38. A pharmaceutical composition comprising a compound of any one ofclaims 1, 16, 20, 26, and 30, and a pharmaceutically acceptable carrier.39. A method for the treatment of inflammation, comprising administeringa therapeutically effective amount of a compound of any one of claims 1,16, 20, 26, and
 30. 40. A method for inhibiting or reducing HIVinfection, comprising administering a therapeutically effective amountof a compound of any one of claims 1, 16, 20, 26, and
 30. 41. A methodfor the treatment of neurodegenerative disease, comprising administeringa therapeutically effective amount of a compound of any one of claims 1,16, 20, 26, and
 30. 42. A method for the treatment of muscle-wastingdiseases, comprising administering a therapeutically effective amount ofa compound of any one of claims 1, 16, 20, 26, and
 30. 43. A method forthe treatment of cancer, comprising administering a therapeuticallyeffective amount of a compound of any one of claims 1, 16, 20, 26, and30.
 44. A method for the treatment of chronic infectious diseases,comprising administering a therapeutically effective amount of acompound of any one of claims 1, 16, 20, 26, and
 30. 45. A method forthe treatment of a hyperproliferative condition, comprisingadministering a therapeutically effective amount of a compound of anyone of claims 1, 16, 20, 26, and
 30. 46. A method for the treatment ofmuscle disuse, comprising administering a therapeutically effectiveamount of a compound of any one of claims 1, 16, 20, 26, and
 30. 47. Amethod for the treatment of immune-related conditions, comprisingadministering a therapeutically effective amount of a compound of anyone of claims 1, 16, 20, 26, and
 30. 48. A method for affecting thelevel of viral gene expression in a subject, comprising administering atherapeutically effective amount of a compound of any one of claims 1,16, 20, 26, and
 30. 49. A method for altering the variety of antigenicpeptides produced by the proteasome in an organism, comprisingadministering therapeutically effective amount of a compound of any oneof claims 1, 16, 20, 26, and 30.